Over the past decade, laser desorption ionization (LDI) techniques, including matrix-assisted laser desorption ionization (MALDI) and surface-enhanced laser desorption ionization (SELDI), have revolutionized the mass spectral analysis of large analytes, including biomolecules. In particular, the recent marriage of LDI sources to a variety of improved tandem (MS/MS) mass spectrometers has significantly expedited protein discovery and characterization.
Certain operational limits currently constrain the usefulness of tandem mass spectrometers in such analyses, however.
For example, collision-induced dissociation (CID) and product ion scans are typically limited to ions having a mass-to-charge (m/z) ratio of no more than about 5000. This limit is attributable in part to constraints on the ability to select precursor ions for CID with adequate resolution, to constraints on the amount of energy that can be delivered to the selected ion, and to stochastic constraints on proton availability to promote fragmentation of the selected ions. This m/z limitation applies to virtually every current tandem mass spectrometer instrument type, including quadrupole time-of-flight (QqTOF), triple quadrupole, ion trap, ion trap time-of-flight, time-of-flight/time-of-flight (TOF/TOF), and ion cyclotron resonance (ICR) mass spectrometers.
The m/z limit for CID and product ion scans particularly constrains tandem MS analysis of singly charged ions, since the unitary charge limits analysis to ions having a mass of no more than about 5000 daltons.
Current MALDI and SELDI ion sources using ultraviolet lasers at 337 nm typically generate a predominance of unit charge molecular ions, often to the exclusion of ions with higher order charge states, thus constraining the ability to use MALDI and SELDI ion sources for tandem MS applications.
Hillencamp et al., U.S. Pat. No. 5,118,937, teach that an increase in incident laser wavelength from 266 nm to greater than about 300 nm, including an increase to IR wavelengths of 3.0–10.6 μm, advantageously decreases analyte absorption of laser irradiation during matrix-assisted laser desorption ionization.
Hillencamp et al. also teach in general terms that certain combinations of IR wavelength and IR matrix produce ions with higher numbers of charges, and notes that such higher order charge states improve collisional and photon bombardment fragmentation in tandem MS applications. The patent does not, however, disclose with specificity which combinations of wavelength and matrix achieve this effect and does not disclose a method for choosing such combinations, although the patent suggests that IR-effective matrices should be chosen to exhibit strong absorption at the chosen wavelength.
There thus exists a need in the art for laser desorption ionization methods that produce a preponderance of gas phase ions having higher order charge states, particularly for use in tandem mass spectral analyses. There also exists a need for methods of choosing combinations of energy absorbing molecules, such as MALDI matrices, and laser wavelengths that lead to a preponderance of ions having higher order charge states during laser desorption ionization of mass spectral analytes.